This invention is directed to a process for using enzymatic glycosylation reactions to prepare polyvalent carbohydrate-containing polymers. These carbohydrate-containing polymers do not cause intolerance reactions in vivo, either in their intact state, or in the form of metabolites produced by physiological degradation processes. The process may also be used for preparing carbohydrate building blocks.
The role of carbohydrates as information carriers in physiologically relevant recognition processes has recently been a subject of intense study. The presence of carbohydrates as ligands on cell surfaces allows them to play, via binding to specific carbohydrate receptors, a crucial part in intercellular communication and in intercellular recognition processes. Carbohydrate ligands on cell surfaces act as recognition elements for, for example, viruses, bacteria, toxins and lectins. Carbohydrates therefore play a crucial role part in, inter alia, bacterial and viral infections, and in the initiation of inflammatory processes such as rheumatoid arthritis, allergies, post-infarct syndrome, shock, stroke and sepsis. Recent investigations have shown that during inflammatory processes the interaction between a carbohydrate ligand and a selectin expressed by endothelial cells mediates adhesion of leukocytes to inflammatory foci. Carbohydrate ligands that are particularly important for cell adhesion are sialylated and/or fucosylated carbohydrates such as sialyl-Lewis X and sialyl-Lewis A.
Therapeutic approaches to treating inflammatory disorders using free oligosaccharides to block the binding of natural ligands to receptors have been largely unsuccessful due to the low affinities observed between the receptor and the oligosaccharide (e.g. the dissociation constant, KDxcx9c10xe2x88x924M for the interaction between a monovalent galactoside and the corresponding lectin, D. T. Connolly et al., J. Biol. Chem. 257, 939, (1982)). These low affinities lead to a requirement for administering very high dosages of carbohydrate. Some divalent structures have, however, been show to have somewhat better binding affinities for a particular receptor. See. for example, Wong et al., J. Amer. Chem. Soc. 115:7549 (1993) and U.S. Pat. No. 5,254,676.
It has been shown that increased interaction between. receptor and ligand can be achieved by coupling a plurality of ligands to a surface. For example, the ligand-receptor interaction between neuraminic acid and the viral protein hemagglutinin is significantly enhanced by coupling multiple carbohydrate moieties to a polymer. Thus the KD for the monovalent interaction is 2xc3x9710xe2x88x924M, and the KD for the polyvalent interaction is 3xc3x9710xe2x88x927M. Spaltenstein et al., J. Amer. Chem. Soc. 113:686 (1991).
Surfaces used to date have been liposomes (Yamazaki, Int. J. Biochem. 24:99 (1991); WO 91/19501; WO 91/19502), polyacrylamides (Rathi et al., J. Polym. Sci.: Part A: Polym. Chem. 29:1895 (1991), Nishimura. et al., Macromolecules 24:4236 (1991)), and polylysine or sulfated polysaccharides. These polyvalent structures have the disadvantage either of having only low stability in vivo or of not being tolerated in vivo due to degradation to toxic metabolites. In the case of polylysine or sulfated polysaccharides non-specific interactions with cell surface structures are also observed. European Published Patent Specifications EP 0 089 938, EP 0 089 939 and EP 0 089 940 describe carbohydrate compounds of varying chain length that are identical to ligands located on cell surfaces or receptors located on microorganisms. These carbohydrate compounds are intended to block receptors located on the microorganisms in vitro and in vivo in order to diagnose and treat diseases. The carbohydrate compounds in these cases may be coupled to a carrier, which may be used, inter alia, to produce antibodies. Similarly, WO 92/02527 discloses an oligosaccharide building block coupled to a solid carrier that may be used to diagnose inflammatory processes. The solid carrier is inert toward physiological systems and is thus not physiologically degraded.
In contrast, EP 0 601 417 A2, which is hereby incorporated by reference in its entirety, discloses a physiologically degradable polymer-based carbohydrate receptor blocker that carries oligosaccharide building blocks on the polymer surface. Improved pharmaceutical activity is obtained by enhanced interaction of the carbohydrate building blocks, which are present polyvalently on the polymer surface, with receptors, and by blocking specific structures. The carbohydrate receptor blocker is physiologically well tolerated, and has a preferred molecular weight of less than about 70 kD.
The physiologically tolerated and physiologically degradable polymer-based carbohydrate receptor blocker disclosed in EP 0 601 417 A2 has the following structure:
carbohydrate side chainsxe2x80x94spacerxe2x80x94hydrophilic biodegradable polymerxe2x80x94potentiator (optional),
where the carbohydrate side chains consist of 1 to naturally occurring, identical or different monosaccharide units that are coupled via one or more bifunctional spacers of natural or synthetic origin to a hydrophilic, biodegradable polymer. The hydrophilic, biodegradable polymer optionally is linked to a potentiator consisting of one or more groups with hydrophobic, hydrophilic or ionic properties, or the potentiator is a crosslinker or enhances solubility.
The carbohydrate portion of the receptor blocker disclosed in EP 0 601 417 A2 may comprise, for example, the following sugar residues:
Galxcex21-4GlcNAc-;
Galxcex21-3GlcNAc-;
SAxcex12-6Galxcex21-4GlcNAc-;
SAxcex12-3Galxcex21-4GlcNAc-;
SAxcex12-3Galxcex21-3GlcNAc-;
Galxcex21-4(Fucxcex11-3)GlcNAc-;
Galxcex21-3(Fucxcex11-3)GlcNAc-;
SAxcex12-3Galxcex21-3(Fucxcex11-4)GlcNAc-;
SAxcex12-3Galxcex21-4(Fucxcex11-3)GlcNAc-.
Other examples of preferred embodiments of the carbohydrate portion are: sialyl-Lewis X, sialyl-Lewis A, VIM-2 and the following blood-group determinants: Lewis A, B, X, Y and A type1, A type2, B type1, B type2 and H type1 and H type2 (see Lemieux, Chem. Soc. Rev., (1978) p. 423 and Chem. Soc. Rev., (1989) p. 347). Particularly preferred embodiments of the carbohydrate portion are sialyl-Lewis X, sialyl-Lewis A or VIM-2.
The formula of sialyl-Lewis X is: NeuNAxcex12-3Gal1-4-(Fucxcex11-3)GlcNAc. The formula of sialyl-Lewis A is: NeuNAxcex12-3Galxcex21-3-(Fucxcex11-4)GlcNAc. The formula of VIM-2 is: NeuNAxcex12-3Galxcex21-4GlcNAcxcex21-3Galxcex21-4(Fucxcex11-3)GlcNAc.
EP 0 601 417 A2 discloses a process for the preparation of the carbohydrate receptor blocker that is suitable for use on a laboratory scale. The carbohydrate receptor blocker accordingly can be synthesized only in milligram to gram quantities. The non-carbohydrate intermediates necessary for the synthesis, i.e., the hydrophilic biodegradable polymer, the bifunctional spacer and the potentiator can, however, be prepared in gram to kilogram amounts.
The limitation in the scale of the overall synthesis of the blocker is due to the synthetic schemes necessary to prepare the carbohydrate portion of the blocker. These schemes can only readily yield amounts of carbohydrate up to one gram. Known synthetic schemes for the preparation of oligosaccharides involve steps that do not proceed with a quantitative yield, and the product mixture obtained after each reaction must be purified by silica gel chromatography. This purification process is generally too costly and elaborate for preparing industrial quantities of material, and is used at the most for purifying final products or valuable intermediates. Additionally, oligosaccharide synthesis frequently uses heavy metal compounds as reagents, which is problematic for subsequent regulatory approval of the blockers as pharmaceutical products, due to possible heavy metal contamination.
In the process described in EP 0 601 417 A2, the required oligosaccharide is linked to the biodegradable polymer by means of a spacer only after the oligosaccharide has been assembled via a large number of chemical and/or chemoenzymatic synthetic stages. These syntheses are very lengthy and difficult because of the well known problems associated with assembly of oligosaccharides, for example, use of protective groups, anomer formation, poor yields of glycosylation reactions, non-stereoselective glycosylation, and the requirement for multiple step reaction schemes.
In view of the problems associated with consecutive reaction and purification steps in the chemical and/or chemoenzymatic synthesis of carbohydrate building blocks, some solid-phase syntheses have recently been proposed. In contrast to the established solid-phase syntheses of oligonucleotides and peptides, the chemical synthesis of oligosaccharides on polymeric solid phases is very difficult owing to the large number of functional groups and the need for stereoselective formation of the glycosidic linkage. Danishefsky et al., (Science, 260:1307 (1993)) linked 3,4-protected glycals via silyl ether linkages to a polystyrene copolymer. The latter is activated as an epoxide and can be linked to further glycal acceptors to give the oligosaccharide. Douglas et al., (J. Am. Chem. Soc. 113:5095 (1991)) described the synthesis of di- and trisaccharides on a PEG-bound glucose unit. Zehavi (J. Am. Chem. Soc. 95:5673 (1973)) used a photosensitive styrene/divinylbenzene copolymer as the polymeric solid phase, where the protected oligosaccharide is cleaved from the polymer by irradiation.
The disadvantages of chemical solid-phase synthesis of oligosaccharides include:
glycosylation reactions are often incomplete;
only a few donor or acceptor glycosylation building blocks are suitable; and
the need for protecting groups appropriate for the particular reaction.
These disadvantages of the chemical synthesis of oligosaccharides on polymeric matrices can be avoided by using enzymatic glycosylation. In this approach reactions take place without protective groups and with stereospecificity. Additionally, enzymatic glycosylation can be employed widely owing to the availability of large number of available glycosyltransferases and nucleotide-activated sugars as glycosyl donors.
As long ago as 1980, Nunez and Barker (Biochemistry 19:499 (1980)) described the enzymatic galactosylation of N-acetylglucosamine linked to agarose via a hexanolamine spacer. Very large amounts of galactosyltransferase enzyme were required, however. Zehavi has described enzymatic galactosylation using galactosyltransferase on photosensitive polymers, where the polymers were either soluble or insoluble in water. Transfer yields were very low, ranging from less than 1% to a maximum of 36% (Zehavi et al., Carbohydrate Res. 124:23 (1983), Zehavi et al., Carbohydrate Res. 128:160 (1984), Zehavi, Reactive Polymers 6:189 (1987), Zehavi et al., Glycoconjugate J. 7:229 (1990) and Zehavi, xe2x80x9cInnovation Perspect. Solid Phase Synthesis Collect. Paperxe2x80x9d, Int. Symp. 1990:389). The resulting disaccharide on the polymer was cleaved from the polymer by the action of light, or by an enzyme (Zehavi et al., Carbohydrate Res. 133:339 (1984)).
Nishimura et al., (Biochem. Biophys. Res. Comm. 199:249-254 (1994)) described the enzymatic preparation of a water-soluble polyacrylamide with 3xe2x80x2-sialyl-N-acetyllactosamine side chains, using stepwise enzymatic glycosylation of a water-soluble, N-acetylglucosamine-carrying polyacrylamide. Only low loading densities were achieved, however, with low overall yields.
A more recent paper by Wong et al., (J. Amer. Chem. Soc. 116:1135 (1994)) describes the enzymatic synthesis of oligosaccharides on a modified silica gel. Owing to the insolubility of the silica gel in the aqueous buffers needed for the enzymatic carbohydrate synthesis and the low loading density of the GlcNAc building block linked via a peptide, only low glycosylation yields were achieved in all three reaction steps. Thus, after enzymatic cleavage of the peptide anchor, product mixtures containing only 20% of the required product and 45% of the precursor were obtained.
WO 92/22661, WO 92/22565 and WO 92/22563 propose enzymatic glycosylations with a sialyltransferase of disaccharides linked to an xe2x80x9cunnatural carrierxe2x80x9d (artificial carrier). An xe2x80x9cunnatural carrierxe2x80x9d is, as a rule, a high or low molecular weight carrier with antigenic properties, for example bovine serum albumin, KLH, HSA, diphtheria or tetanus toxin, etc., or a solid carrier that is inert toward physiological systems.
It is apparent, therefore, that an efficient method for preparing a polyvalent, physiologically tolerated and physiologically degradable carbohydrate-containing polymer is greatly to be desired. In particular, it is greatly desirable to synthesize the carbohydrate-containing polymer by high yield enzymatic glycosylation reactions.
It is therefore an object of the present invention to provide a process for preparing polyvalent carbohydrate-containing polymers that are physiologically degradable in vivo. The polyvalent carbohydrate-containing polymers thus prepared comprise a hydrophilic, biodegradable polymer unit, a disaccharide or oligosaccharide unit, and a bifunctional spacer linking the disaccharide or oligosaccharide units to the biodegradable polymer unit.
It is another object of the invention to provide a process for the diagnosis, therapy or prophylactic treatment of bacterial or viral infections, post-infarct syndrome, shock, stroke, acute and chronic organ rejection, vasculitis, inflammatory disorders, rheumatoid arthritis, metastatic tumors and shock lung, comprising the use of a carbohydrate-containing polymer that is physiologically degradable in vivo, where the carbohydrate-containing polymer comprises a hydrophilic, biodegradable polymer unit, a disaccharide or oligosaccharide unit, and a bifunctional spacer linking the disaccharide or oligosaccharide units to the biodegradable polymer unit.
It is yet another object of the invention to use the carbohydrate-containing polymer prepared according to the methods of the invention for preparing free oligosaccharides.
In accomplishing the foregoing objects of the invention, there has been provided, in accordance with one aspect of the current invention, a process to assemble the carbohydrate portion of the carbohydrate-containing polymer by enzymatic glycosylation reactions in aqueous buffer systems in a homogeneous phase directly onto a biodegradable polymer. The yields of the glycosylation reactions are greatly improved in comparison with the yields of known processes. The process comprises the steps of: (a) covalently linking a monosaccharide or oligosaccharide unit to a spacer to form a monosaccharide or oligosaccharide-spacer complex; (b) covalently linking the monosaccharide or oligosaccharide-spacer complex to a hydrophilic, biodegradable polymer unit to form an acceptor unit; and (c) coupling a monosaccharide donor unit to the acceptor unit by enzymatic glycosylation. In accordance with another aspect of the invention the process comprises the steps of: (a) covalently linking the hydrophilic, biodegradable polymer unit to the spacer to form a biodegradable polymer-spacer complex; (b) covalently linking the biodegradable polymer-spacer complex to a monosaccharide or oligosaccharide unit to form an acceptor unit; and (c) coupling a monosaccharide donor unit to the acceptor unit by enzymatic glycosylation.
In accordance with a further aspect of the invention, there has been provided a process where the enzymatic glycosylation of the acceptor unit takes place in a homogeneous aqueous phase, preferably using nucleotide-activated carbohydrates as the monosaccharide donor units. In another preferred embodiment the glycosylation reaction is catalyzed by at least one glycosyltransferase, at between about pH 6.0 and about pH 8.5. Preferably about 0.01 to 10 units of the glycosyltransferase are used, and the enzymatic glycosylation is carried out at a temperature between about 10xc2x0 C. and about 40xc2x0 C. for between about 1 to about 5 days.
In accordance with another aspect of the invention, alkaline phosphatase is added to the reaction medium when the monosaccharide donor unit is added in equimolar amount or in excess.
In accordance with still another aspect of the invention, the hydrophilic biodegradable polymer unit is selected from the group consisting of a polycarbonate, polyester, polyamide, polyanhydride, polyiminocarbonate, polyorthoester, polydioxanone, polyphosphazene, polyhydroxycarboxylic acid, polyamino-acid and a polysaccharide. The polymer preferably is a polyamino-acid having a molecular weight up to about 70 kD, wherein the polyamino-acid is in polyamide or polyanhydride form. The polyamino-acid is preferably selected from the group consisting of poly-xcex1,xcex2-(2-hydroxyethyl)-D,L-aspartamide, poly-D,L-succinimide, polyglutamate, poly-L-lysine methyl ester fumaramide and a copolymer of these polyamino-acids.
In accordance with a still further aspect of the invention, the acceptor unit has the formula: (monosaccharide or oligosaccharide)xe2x80x94Oxe2x80x94{Q1xe2x80x94(CH2)p-Q2}r(polymer unit), wherein Q1 is xe2x80x94CH2 or xe2x80x94COxe2x80x94, Q2 is xe2x80x94NH or xe2x80x94COxe2x80x94NHxe2x80x94, p is 1-6, and r is 1 or 2. The linkages connecting the spacer to the monosaccharide or oligosaccharide unit and to the biodegradable polymer unit are preferably formed by reactions selected from the group consisting of alkylation, reductive alkylation, acylation and addition onto a double bond.
In accordance with yet another aspect of the invention, the disaccharide or oligosaccharide unit of the carbohydrate-containing polymer is selected from the group consisting of: SAxcex12-6Galxcex21-4GlcNAc-; SAxcex12-3Galxcex21-4GlcNAc-; SAxcex12-3Galxcex21-3GlcNAc-; Galxcex21-4(Fucxcex11-3)GlcNAc-; Galxcex21-3(Fucxcex11-3)GlcNAc-; SAxcex12-3Galxcex21-3(Fucxcex11-4)GlcNAc-; SAxcex12-3Galxcex21-4(Fucxcex11-3)GlcNAc-, sialyl-Lewis X, sialyl-Lewis A, VIM-2, Lewis A, Lewis B, Lewis X, Lewis Y, Lewis A type1, Lewis A type2, Lewis B type1, Lewis B type2, Lewis H type1 and Lewis H type2.
In accordance with a still further aspect of the invention, the nucleotide-activated carbohydrate is preferably selected from the group consisting of: UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetyl-galactosamine, UDP-glucuronic acid, CMP-neuraminic acid, GDP-fucose, GDP-mannose, dTDP-glucose and dUDP-galactose. The glycosyltransferase is preferably selected from the group consisting of: xcex2-1,4-galactosyltransferase, Gal-xcex2-1-4-GlcNAc xcex1-2-6-sialyltransferase, Gal-xcex2-1-3-GalNAc xcex1-2-3-sialyltransferase, Gal-xcex2-1-3(4)-GlcNAc xcex1-2-3-sialyltransferase, GalNAc xcex1-2-6-sialyltransferase, N-acetylglucosaminytransferases, xcex1-1-3-fucosyltransferase, xcex1-1-2-fucosyltransferase, xcex1-3/4-fucosyltransferase, and xcex1-1-2-mannosyltransferase. The disaccharide or oligosaccharide unit preferably contains 2-20 monosaccharide units.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The present invention provides a process for preparing polyvalent, physiologically tolerated and physiologically degradable carbohydrate-containing polymers that act as receptor blockers. The process is distinguished by using enzymatic glycosylation reactions to assemble the carbohydrate portion of the receptor blocker. These reactions take place directly on the soluble biodegradable polymer in a homogeneous phase in aqueous buffer systems. The yields of the glycosylation reaction are greatly improved in comparison with the yields of known processes and, as a rule, take place quantitatively. The loading densities of oligosaccharide on the polymer are also greatly increased over those achieved in the prior art. The carbohydrate-containing polymers prepared according to the methods of the invention may also be used for preparing the free oligosaccharides.
The physiologically tolerated and physiologically degradable carbohydrate-containing polymer comprises:
a) a hydrophilic, biodegradable polymer unit;
b) at least one di- or oligosaccharide unit; and
c) at least one bifunctional spacer by which the di- or oligosaccharide units are linked to the polymer unit.
The carbohydrate-containing polymer is prepared by a process wherein an acceptor is prepared by covalently linking together a mono- or oligosaccharide, a spacer and a hydrophilic biodegradable polymer, followed by attaching one or more additional monosaccharide units by enzymatic glycosylation. The spacer can first be linked to the mono- or oligosaccharide, followed by coupling to the polymer, or the spacer can first be linked to the polymer followed by coupling to the mono- or oligosaccharide. The enzymatic glycosylation reaction takes place stereoselectively and with surprisingly high yields directly on the polymer and can, because each glycosylation step takes place essentially quantitatively, be repeated as often as necessary with any desired donors. The process preferably uses nucleotide-activated carbohydrates as donors and one or more glycosyltransferases as the enzyme in the glycosylation reactions.
The aqueous medium is preferably a buffer system suited to the particular glycosyltransferase, and the buffer system preferably has a concentration of 0.01 M to 1 M. The buffer preferably contains any cations necessary for activating the particular glycosyl-transferase, for example, Mn2+. The pH of the glycosylation reaction is preferably between about 6.0 and about 8.5, more preferably between about 6.5 and about 8.0, and most preferably between about 7.0 and about 7.5.
When the donor is added in approximately equimolar amounts or in excess, alkaline phosphatase is preferably added to the reaction medium. Between about 0.01 to about 10 units of the glycosyltransferase are added to the reaction mixture. The glycosylation is carried out at a temperature between about 10xc2x0 C. to about 40xc2x0 C., preferably at about 20xc2x0 C. to about 37xc2x0 C., even more preferably at about 25xc2x0 C. to about 37xc2x0 C., for about 1 to 5 days.
The invention is explained in detail hereinafter:
The acceptor for the enzymatic glycosylation reaction consists of a mono- or oligosaccharide which is covalently linked via a spacer to a biodegradable hydrophilic polymer. The polymer can be provided with a potentiator comprising one or more groups with hydrophobic, hydrophilic or ionic properties, or the potentiator is a crosslinker or enhances solubility. The acceptor is synthesized by methods known to the skilled artisan. The individual building blocks of the acceptor are described hereinafter.
A. Biodegradable Hydrophilic Polymer
By definition, the polymer consists of at least two identical or different monomer units that are linked together in linear or branched fashion and that may display a molecular weight distribution. The polymer is preferably a polyamino-acid linked via polyamide or anhydride linkages, with a molecular weight less than or equal to about 70 kD. The preferred minimum size of the polymer is about 2 kD in order to achieve increased residence time in the blood by comparison with low molecular weight carriers.
Preferred polyamino-acids for preparing carbohydrate-containing polymers are polyaspartamides, polysuccinimides, polyglutamates and polylysine-fumaramides. Particularly preferred polymers include, for example, poly-xcex1,xcex2-(2-hydroxyethyl)-D,L-aspartamide, poly-D,L-succinimide, polyglutamate, poly-L-lysine methyl ester furamamide, and copolymers thereof.
The biodegradable, hydrophilic polymer is prepared by processes known to the skilled artisan. Suitable methods are described, for example, in Elias, Makromolekxc3xc[Macromolecules], Volumes 1 and 2, Hxc3xcthig and Wepf Verlag, Basle, Switzerland, 1991/92 and Braun et al., Praktikum der Makromolekularen organischen Chemie [Practical Molecular Organic Chemistry], Hxc3xcthig Verlag 1979.
Thus, for example, poly-D,L-succinimide (PSI) is prepared by the method of Neri et al., (J. Med. Chem., 16:893 (1973)) by the action of 85% phosphoric acid on aspartic acid at temperatures of 160xc2x0 C.-180xc2x0 C. Reaction of PSI polymer with hydroxyethylamine at room temperature or slightly elevated temperature results in poly-xcex1,xcex2-(2-hydroxyethyl)-D,L-aspartamide (PHEA). Alcohol groups on the PHEA can be esterified by well known methods. See, for example, U.S. Pat. No. 5,041,291. Partial reaction of PSI with ethanolamine results in corresponding copolymers. See U.S. Pat. No. 5,229,469. Basic hydrolysis of PSI leads to polyaspartic acid (in an analogous manner to that described by Giammona et al., (Chem Pharm. Bull. 37:2245 (1989)). In similar fashion to the reaction with hydroxyethylamine, PSI can also be reacted with other amines (see EP 0 548 794), which makes it possible to introduce additional functional groups which may act as potentiators.
Poly-L-lysine methyl ester fumaramide, can be prepared by boundary phase polycondensation of L-lysine methyl ester and fumaryl chloride. See U.S. Pat. No. 4,835,248. The methyl ester groups can be reacted directly or after partial hydrolysis and subsequent activation, for example as p-nitrophenyl ester, with the mono-, di- and oligosaccharides containing amino groups.
In an analogous way, i.e., using p-nitrophenyl esters, it is possible to prepare carbohydrate-containing polymer based on polyglutamates. See Anderson in xe2x80x9cMacromolecules as Drugs and as Carriers for Biologically active Materialsxe2x80x9d (Ed: D. A. Tirell), NY Acad. Sci., NY, 1985, pp. 67-75.
B. Spacer
The covalent linkages between (i) the polymer and the spacer; (ii) the polymer and the covalently linked spacer/carbohydrate compound; (iii) the spacer and the covalently-linked polymer/potentiator, and (iv) the covalently-linked polymer/potentiator and the covalently linked spacer/carbohydrate compound, are formed by reaction between a reactive group and an activated group. Suitable reactions are well known to the skilled artisan, and include alkylation, reductive alkylation, acylation or addition onto a double bond. These and other suitable reactions are described in Larock, Comprehensive Organic Transformations, 1989, VCH Verlagsgesellschaft Weinheim).
The reactive group may be located at the terminus of the spacer or at the terminus of the covalently-linked spacer/carbohydrate compound. The activated group may be located on the polymer or on the covalently-linked polymer/potentiator compound. The activated group may be located at the end of the spacer or at the terminus of the covalently-linked spacer/carbohydrate compound. The reactive group may be located either on the polymer or on a covalently-linked polymer/potentiator compound.
The spacer preferably has the formula I:
(mono- or oligosaccharide)xe2x80x94Oxe2x80x94[Q1xe2x80x94(CH2)pxe2x80x94Q2]rxe2x80x94(polymer)xe2x80x83xe2x80x83I,
wherein
Q1 is xe2x80x94CH2 or xe2x80x94COxe2x80x94,
Q2 is xe2x80x94NH or xe2x80x94COxe2x80x94NHxe2x80x94,
p is an integer from 1 to 6 and
r is 1 or 2
C. Carbohydrate Portion of the Acceptor
The carbohydrate portion of the acceptor for the enzymatic glycosylation reaction may be derived from natural sources or may be prepared chemically, chemoenzymatically, or enzymatically. Suitable natural sources for carbohydrates are well known to the skilled artisan. Processes for purifying oligosaccharides are also well known to the skilled worker.
Methods for chemical, enzymatic or chemoenzymatic synthesis of carbohydrates that bind to cell surface receptors are similarly known to one of skill in the art. Many suitable chemical syntheses are described, for example, in: Carbohydrate Research, Elsevier Science Publishers B.U. Amsterdam; Journal of Carbohydrate Chemistry, Marcel Dekker Inc. New York; Paulsen, Angew. Chem. 94:184 (1982) and 102:851 (1990); Schmidt Angew. Chem. 98:213 (1987); and Kunz. Angew. Chem. 98:247 (1987). Enzymatic syntheses are described in, for example: Carbohydrate Synthesis, ACS Symposium Series 466 (1991); Nilsson, Applied Biocatalysis 1991:117; David et al., Adv. Carbohydr. Chem. Biochem. 49:175 (1991); Ichikawa et al., Anal. Biochem. 202:215 (1992); Drueckhammer et al., Synthesis 1991:499; Toone et al., Tetrahedron 45:5365 (1980).
Mono- or oligosaccharides prepared in this way can be prepared either with a free reducing end or in a spacer-linked form. The spacer is introduced by processes known to the skilled worker for chemical or enzymatic glycosylation.
The acceptor for the enzymatic glycosylation reaction, prepared as described supra, consists of a mono- or oligosaccharide covalently linked via a spacer to a biodegradable hydrophilic polymer. The polymer may optionally be provided with a potentiator. Enzymatic glycosylation of the acceptor preferably takes place in homogeneous aqueous phase, preferably using nucleotide-activated carbohydrates as donors, and glycosyltransferases as enzymes.
The acceptor is dissolved in an aqueous buffer system. The buffer is chosen as appropriate for the particular glycosyltransferase and may contain, for example, cacodylate, HEPES, PIPES, MOPS, citrate, bicarbonate, etc., at concentrations of about 0.01 M to 1 M. The buffer also contains any cations necessary for activating the particular glycosyltransferase, for example Mn2+. The pH of the buffer is also chosen to be appropriate for a particular glycosyltransferase, and is preferably between about 6.0 and about 8.5, more preferably between about 6.5 and about 7.8, and most preferably between about 7.0 and about 7.5.
After the acceptor has been dissolved in the aqueous buffer system, the donor is added. The donor moiety is a nucleotide-activated sugar or an analog of a nucleotide-activated sugar, for example d-UDP-Glc, d-UDP-Gal, d-UDP-GlcNAc, where d-UDP is deoxyuridine diphosphate. The nucleotide-activated sugars are commercially available, for example from Sigma (St. Louis, Mo.), Boehringer Mannheim (Indianapolis, Ind.), and Genzyme (Cambridge, Mass.), or may prepared by well-known methods using chemical or enzymatic syntheses, or may be isolated from natural sources. Nucleotide-activated sugar analogs may also be prepared by the same methods. The donor is added in a 1.1 to 2 fold excess, or is regenerated in situ by known processes. See, for example: Ichikawa et al. J. Amer. Chem. Soc. 114:9283 (1992); Wong et al., J. Org. Chem. 57:4343 (1992); Ichikawa et al., J. Amer. Chem. Soc. 113:6300 (1991); and Wong et al., J. Org. Chem. 47:5416 (1982).
A common donor used in the enzymatic galactosylation is UDP-galactose. UDP-glucose may also be enzymatically epimerized in situ using the enzyme UDP-galactose 4-epimerase to form UDP-galactose (J. Thiem et al., Angew. Chem. 102, 78 (1990)). When the donor is used in stoichiometric amounts or in excess in the enzymatic glycosylation, it is necessary to decompose enzymatically the UDP liberated in the reaction using alkaline phosphatase, in order to prevent inhibition of the glycosyltransferase. See Unverzagt et al., J. Amer. Chem. Soc. 112:9308 (1990).
Other examples of nucleotide-activated sugars that may be used in the glycosylation include UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetyl-galactosamine, UDP-glucuronic acid, CMP-neuraminic acid, GDP-fucose, GDP-mannose, dTDP-glucose and dUDP-galactose. Other UDP-activated sugars suitable for use in glycosylation reactions will be apparent to those of skill in the art. Processes known to the skilled worker for the preparation of nucleotide-activated sugars are described, for example in: Kittelmann et al., Ann. N.Y. Acad. Sci. 672:444 (Enzyme Engineering) (1992); Makino et al., Tetrahedron Lett. 34:2775 (1993); Martin et al., Tetrahedron Lett. 34:1765 (1993); European Patent Application 0 524 143 A1; Ikeda, Carbohydrate Res. 224:123 (1992); Kean Glycobiology 1:441 (1991); Ichikawa et al., T. Org. Chem. 57:2943 (1992); Adelhorst et al., J. Org. Chem. 57:2943 (1992); Adelhorst et al., Carbohydrate Research 242:69 (1993); Schmidt et al., Liebigs Ann. Chem. 1991:121; Stiller et al., Liebigs Ann. Chem. 1992:467; Heidlas et al., J. org. Chem. 57:146 (1992), Heidlas, Acc. Chem. Res. 25:307 (1992); Simon et al., J. Org. Chem. 55:1834 (1990); Wong et al. J. Org. Chem. 57:4343 (1992); and Pallanca et al., J. Chem. Soc. Perkin Trans. I 1993:3017.
The glycosylation reaction is carried out by adding about 0.01 to about 10 units of an appropriate glycosyltransferase are added to a solution of the acceptor the nucleotide-activated sugar dissolved in the aqueous buffer system. The glycosyltransferases are commercially available, for example from Sigma (St. Louis, Mo.), Boehringer Mannheim (Indianapolis, Ind.), and Genzyme (Cambridge, Mass.), or may be isolated from natural sources, or may be prepared by recombinant DNA technology. Examples of glycosyltransferases which may be used in the enzymatic glycosylation include:
xcex2-1,4-galactosyltransferase [Barker et al., J. Biol. Chem. 247:7135 (1972) and Krezhorn et al., Eur. J. Biochem. 212:113 (1993)];
Gal-xcex2-1-4-GlcNAcxcex1-2-6-sialyltransferase [Paulsonet al., J. Biol. Chem. 252:2363 (1977); Higa et al., J. Biol. Chem. 260:8838 (1985); and Weinstein et al., J. Biol. Chem. 257:13835 (1982)];
Gal-xcex2-1-3-GalNAc xcex1-2-3-sialyltransferase [Gillespie et al., J. Biol. Chem. 267:21004 (1992)];
Gal-xcex2-1-3(4)-GlcNAc xcex1-2-3-sialyltransferase [Weinstein et al., J. Biol. Chem. 257:13835 (1982) and Nemansky et al., Glycoconjugate J. 10:99 (1993)];
GalNAc xcex1-2-6-sialyltransferase [Gros et al., Biochemistry 28:7386 (1989);
N-acetylglucosaminytransferases [Oehrlein et al., Carbohydrate Res. 244:149 (1993); Szumilo et al., Biochemistry 26:5498 (1987); Hindsgaul et al., J. Biol. Chem. 266:17858 (1991); and Look et al., J. Org. Chem. 58:4326 (1993)];
xcex1-1-3-fucosyltransferase [Weston, J. Biol. Chem. 267:4152 (1992)];
xcex1-1-2-fucosyltransferase [Beyer, J. Bidl. Chem. 255:5364 (1980)];
xcex1-3-4-fucosyltransferase [Johnson, Glycoconjugate J. 9.241 (1992)];
xcex1-1-2-mannosyltransferase [Wang, J. Org. Chem. 58:3985 (1993)]; and
generally: Beyer et al., Advances in Enzymology 52:23 (1981) and WO 93/13198.
The enzymatic glycosylation is carried out at a temperature between about 10xc2x0 C. to about 40xc2x0 C., preferably at about 20xc2x0 C. to about 37xc2x0 C., particularly preferably at about 25xc2x0 C. to 37xc2x0 C., for 1 to 5 days. Once reaction is complete, as indicated by standard chromatography methods such as HPLC or TLC, the reaction is worked up by dialyzing against double-distilled water. The carbohydrate-containing polymer may subsequently be further purified by chromatography methods such as, for example, size exclusion chromatography.
The process according to the invention is particularly suitable for preparing carbohydrate-containing polymer with the following oligo- or disaccharide units:
Galxcex21-4GlcNAc-;
Galxcex21-3GlcNAc-,
SAxcex12-6Galxcex21-4GlcNAc-;
SAxcex12-3Galxcex21-4GlcNAc-;
SAxcex12-3Galxcex21-3GlcNAc-;
Galxcex21-4(Fucxcex11-3)GlcNAc-;
Galxcex21-3(Fucxcex11-3)GlcNAc-;
SAxcex12-3Galxcex21-3(Fucxcex11-4)GlcNAc-;
SAxcex12-3Galxcex21-4(Fucxcex11-3)GlcNAc-;
sialyl-Lewis X, sialyl-Lewis A, VIM-2 and the following blood-group determinants Lewis A, B, X, Y and A type1, A type2, B type1, B type2 and H type1, H type2. See, Lemieux, Chem. Soc. Rev., 1978:423 and Chem. Soc. Rev. 1989:347.